To initiate an adaptive immune response, antigen presenting cells (APCs) must process “foreign” proteins into peptides. These peptides associate with MHC proteins which transport these peptides to the APCs' plasma membrane where they are recognized in the context of MHC proteins by helper and cytotoxic T-cell precursors. Helper T-lymphocyte precursors recognize peptide in association with Class II MHC proteins while cytotoxic T-lymphocyte (CTL) precursors recognize peptide in association with Class I MHC proteins.
The major types of APC's (mononuclear phagocytes and dendritic cells) express plasma membrane receptors for ATP4− (1,2,3). These receptors are called P2X7 receptors. Binding of ATP4− to P2X7 receptors opens a “pore” in the plasma membranes of macrophages (4), and of dendritic cells (3,5) that allows molecules of up to ˜900 daltons M.W. into the cytoplasm of these cells without killing the cells. The ATP4−-activated pore of macrophages was first identified by applicants. The P2X7 receptor is formed by the association of multiple protein subunits each 595 aa long.
At neutral pH and in the presence of physiological salts most of the ATP in extracellular fluids is complexed with divalent cations, primarily Mg2+ and Ca2+. Under these conditions, the equilibrium between MgATP2−/CaATP2− and ATP4− strongly favors MgATP2−/CaATP2−. Consequently, [MgATP2−/CaATP2−] in excess of 3 mM are required to achieve an [ATP4−] of >130 μM, the [ATP4−] needed to induce pore formation by P2X7 receptors (4). [ATP]>3 mM are rarely if ever found in extracellular fluids under physiological conditions. However, apoptotic cells contain >5 mM ATP (6).
Scavenger receptors present on the plasma membranes of APCs promote the phagocytosis of apoptotic cells. Following their ingestion, apoptotic cells are sequestered and lysed within phagolysosomes of these APCs. This releases both ATP and various peptides into the vacuole of the the APCs' phago-lysosome. It is hypnothesized that the ATP released from apoptotic cells into phagolysosomes of APCs opens P2X7 receptors. This provides a pathway by which potentially immunogenic peptides from “foreign” apoptotic cells, and potentially “toleragenic” peptides from self apoptotic cells, enter the cytoplasm of APCs. These peptides then can be carried by TAP proteins into the endoplasmic reticulum where they associate with Class I MHC proteins. APCs and especially immature dendritic cells (1), recycle Class II MHC molecules from their phago-lysosomes to the plasma membrane. Thus peptide antigens released into phago-lysosomes are efficiently presented in association with Class II MHC proteins.
Antigen presenting cells (APCs) whose Class I and Class II MHC molecules contain antigen peptides elicit cytotoxic and helper T-lymphocytes. In some instances, these cytotoxic and helper lymphocytes cause tumor regression. Devised herein is a novel method for delivery of immunogenic peptides to macrophages and dendritic cells for presentation by Class I and Class II MHC proteins. The method uses as a delivery vehicle IgG-opsonized resealed red blood cell ghosts (rRBCg) containing immunogenic peptides for delivery to Class II MHC proteins, and IgG-opsonized-rRBCg containing immunogenic peptides and ATP for delivery to Class I MHC proteins. In the latter instance, the method makes use of ATP4−-activated receptors (which may be P2X7 or other ATP receptors) present in phagolysosomal vesicles to deliver immunogenic peptides to the cytoplasmic matrix of APCs (i.e., dendritic cells and macrophages).
Human red blood cell ghosts or other particles that can be filled with antigens (e.g., liposomes) and coated with ligands (IgG, oxidized lipids, sugars, polyanions), for receptors on antigen presenting cells (e.g., dendritic cells, Langerhans cells, monocytes, macrophages), are used as vehicles to encapsulate antigens (e.g., peptides, carbohydrates lipids, glycoproteins, glycolipids, lipoproteins), and adenosine triphosphate (ATP) or other ligands for ATP receptors (e.g. P2X7 and other ATP receptors)]. The antigens may be an antigen derived from, and/or induce immune responses that affect microbial pathogens, tumor cells, and/or immuno-regulatory pathways. Ligands on the particle will promote ingestion of the particle by antigen presenting cells. Enzymes released into particle-containing phagosomes of antigen presenting cells will lyse the particle, releasing ATP and/or other substances that activate ATP receptors (such as P2X7 receptors, but not limited to these receptors) into these phagosomes. Activation of the receptors will create “pores” in the phagosomes' membranes through which antigens (e.g., antigenic peptides, carbohydrates, lipids) can enter the cytoplasm for processing and presentation to T-cells in association with conventional Class I MHC molecules, or other antigen presenting receptors.
The invention disclosed herein is useful as a vaccine, as a method for delivery of antigens to the cytoplasmic matrix of antigen presenting cells to induce immunity, to activate cytotoxic effects against tumor cells, and/or to suppress immunity/induce tolerance. The delivery system may also be used to deliver Th1 stimulatory cytokines (e.g., I1–I2, interfereon gamma) along with the antigen. The invention provided herein is a simple delivery system for purified antigens or crude cell extracts directly into the cytoplasmic matrix of antigen presenting cells for presentation by class I or II MHC and provides the advantage of not requiring isolation of host antigen presenting cells.